The invention relates generally to methods and compositions for culturing human pluripotent stem cells, and, more particularly, to methods and compositions having thermostable fibroblast growth factor (FGF) proteins for improved culture efficiency.
Human pluripotent cells, such as human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells have the potential to proliferate indefinitely and to differentiate into cells of all three germ layers (Lowry et al., PNAS 105: 2883-2888, 2008; Park et al., Nature 451:141-U141, 2008; Reubinoff et al., Nat. Biotechnol. 18:399-404, 2000; Takahashi et al., Cell 131:861, 872, 2007; Thomson et al., Science 282:1145-1147, 1998; Yu et al., Science 318:1917-1920, 2007). These properties make human pluripotent cells invaluable for studying embryogenesis, for drug discovery, and for clinical applications.
Current in vitro culture methods for human ES and iPS cells require the addition of exogenous growth factors (Amit et al., Nat. Rev. Drug Discov. 8:235-253, 2004; Ludwig et al., Nat. Biotechnol. 24:185-187, 2006; Sato et al., Nat. Med. 10:55-63, 2004; Vallier et al., J. Cell Sci. 118:4495-4509, 2005; Wang et al., Blood 110:4111-4119, 2007). It is presently thought that three growth factors are sufficient to maintain pluripotency and self-renewal of human ES and iPS cells through activation of the FGF, TGF/Nodal, and Insulin/IGF pathways (Bendall et al., Nature 448:1015-1021 (2007); Eiselleova et al., Stem Cells 27:1847-1857 (2009); Vallier et al., J. Cell Sci. 118:4495-4509 (2005)).
The FGF pathway has been implicated in many stages of human pluripotent cell regulation, cell survival, proliferation, pluripotency, and lineage determination during differentiation (Eiselleova et al., Stem Cells 27:1847-1857, 2009; Lanner and Rossant, Development 137:3351-3360, 2010; Levenstein et al., Stem Cells 24:568-574, 2006; Vallier et al., J. Cell Sci. 118:4495-4509, 2005; Xu et al., Nat. Meth. 2:185-190, 2005). The FGF pathway is activated through the binding of FGF proteins to FGF receptors, which triggers MAP kinase cascades to regulate downstream events (Lanner and Rossant, 2010).
FGF-1-9 are 150-250 amino acid proteins with approximately 30-70% sequence homology in their 120-amino acid core region (Ornitz et al., Genome Biol. 2:3005.1-3005.12 (2001); Itoh et al., Trends Genet. 20:563-569 (2004)). Because of their substantial sequence homology, new members of the FGF family were identified in several species, from Caenorhabditis elegans to Homo sapiens (Itoh et al.), using homology-based methods. Twenty-two FGF family members have been identified in humans and mice (Ornitz et al., 2001; Itoh et al., 2004).
While different FGF proteins are used for various applications in cell culture, qualitative differences in cell responses elicited by the various FGF proteins remain ill-defined and poorly understood. The functional difference between FGF proteins that can and cannot support human pluripotent stem cells might be attributable to (1) the different affinity of the various FGF proteins to each of the four FGF receptors (FGFR) that lead to the activation of specific pathways (Eswarakumar et al., Cytokine Growth Factor Rev. 16:139-149, 2005; Mohammadi et al., Cytokine Growth Factor Rev. 16:107-137, 2005; Zhang et al., J. Biol. Chem. 281:15694-15700, 2006); and (2) the differential expression of FGFs and FGFRs in specific tissues (Beenken and Mohammadi, Nat. Rev. Drug Discov. 8:235-253, 2009). However, these factors insufficiently explain the functional differences between FGF-2 and other FGF proteins in human ES cell culture.
FGF-2 is routinely used for human ES and iPS cell culture (Levenstein et al., Stem Cells 24:568-574, 2006). Interestingly, FGF-1 does not support hESC pluripotency or cell survival, even though FGF-1 targets the same set of receptors as FGF-2 (Zhang et al., J. Biol. Chem. 281:15694-15700, 2006).
While FGF-2 supports pluripotency in defined long-term human pluripotent cell cultures, high FGF-2 concentrations (e.g., 100 ng/ml) are required, which significantly increases culture cost. It has been suggested that high FGF-2 concentrations might be required to satisfy specific dose-dependent signaling thresholds, and to overcome obstacles such as protein degradation (Levenstein et al., Stem Cells 24:568-574, 2006). Heparin and heparan sulfate can facilitate binding between FGF and FGFR to stimulate downstream activation (Levenstein et al., Stem Cells 26:3099-3107, 2008; Mohammadi et al., Curr. Opin. Struct. Biol. 15:506-516, 2005). Heparin and heparan sulfate promote pluripotency (Fume et al., PNAS 105:13409-13414, 2008; Levenstein et al., Stem Cells 26:3099-3107, 2008), although it is unclear whether they do so via the FGF pathway. Heparin appears to increase the stability of FGF-1 and might be important in the formation of FGF-1-FGFR complexes (Zakrzewska et al., J. Biol. Chem. 284:25388-25403 (2009)). While FGF-2 from zebrafish is capable of supporting self-renewal (Ludwig et al., Nat. Meth. 3:637-646, 2006), effective mammalian FGFs that can be used as an alternative to mammalian wild type FGF-2 are not known. Thus, there is a need in the art for more efficient growth factors that can support human pluripotent stem cells in culture.